Microglia serve as resident immune cells in the brain and are considered key contributors to neuroinflammation, a process that has been implicated in the negative, long-term effects of alcohol on the central nervous system. The molecular mechanisms underlying ethanol-induced microglial activation are still unclear and the role of epigenetic regulation in this process is essentially unknown. Preliminary data generated by our labs indicate that one-carbon metabolism as well as enzymes involved in histone methylation are altered in microglia after acute ethanol exposure. Additionally, we have demonstrated that histone methylation changes occur in vitro and in vivo in microglia after ethanol exposure and knockdown of a specific histone demethylase can affect the pro-inflammatory response of microglia. We hypothesize that methylation is an important epigenetic modification that influences partial activation of microglia after acute ethanol exposure and that indirect ?classical? activation mediated by methylation occurs in response to ethanol-induced neuronal damage as a result of chronic exposure. In order to test our hypothesis, we plan to 1) characterize ethanol- induced changes in the microglial histone methylation code in both primary pure cultures of microglia and microglia-neuron co-cultures and 2) determine the functional impact of histone methylation in ethanol-induced microglial activation in mouse models of short- and long-term ethanol exposure. The proposed studies incorporate novel approaches such as activity-based protein profiling, methylation-specific quantitative mass spectrometry and ChIP-Seq in order to carry out these aims. This project will be the first ever comprehensive global-scale analysis of methylation and its regulators, which could determine, at least partly, the epigenetic code related to microglial activation phenotype. Characterization of these ethanol-responsive pathways in microglia could also lead to further insight into the development of novel epigenetic therapies for the treatment of neuroinflammation resulting from long-term alcohol abuse.